1a. Objectives (from AD-416):
1. Detection and control of fungi in post-harvest, stored corn.
2. Evaluate commercially available plant compounds for their in vitro activity against agriculturally important bacteria and fungi.

1b. Approach (from AD-416):
This project investigates new technologies to detect Aspergillus (A.) flavus and Fusarium (F.) verticillioides growth on corn and novel methods to inhibit their growth and mycotoxin production. Secondary metabolic volatiles unique for the growth of toxigenic strains of the fungi will be identified and shared with our stakeholder who will develop electronic, “real-time” sensors to detect the growth of these fungi. Safe, commercially available plant antifungal volatiles will be tested for their in vitro activity against these fungi. Promising compounds will be further tested to determine their ability to prevent fungal growth in small-scale, bench models of stored corn. These same compounds in the liquid form will be studied for their in vitro activity against the bacterium, Leuconostoc (L.) mesenteroides (a problem in sugar cane factories) and the fungi Penicillium (P.) digitatum and P. italicum which cause post harvest citrus rot. ARS collaborators will use this data to develop novel technologies to prevent the growth of these fungi in raw sugar cane juice and stored oranges.

3. Progress Report:
Progress was made on all planned research. Research was completed on the identification of unique secondary metabolic volatiles produced by toxigenic strains of Aspergillus (A.) flavus, as compared to non-toxigenic strains and corn controls, when grown separately on sterile and non-sterile corn. During FY 2011, results were reported to our stakeholder, Sensor Development Corporation, which used this data to develop a real-time sensor which is undergoing Beta testing by a large grain company in a silo. Later in 2012, work will commence on the study of unique secondary metabolic volatiles produced by the fungus Fusarium (F.) verticillioides on sterile corn. Investigations continue on the antifungal and antibacterial properties of safe, inexpensive plant compounds, especially those with volatile properties, and specifically those having significant activity at low concentrations (= 20 µM) against fungi that produce toxins on corn. These fungi include A. flavus, F. verticillioides. Other fungi targeted include F. graminearum (produces mycotoxins on wheat), and Penicillium (P.) digitatum and P. italicum, both of which cause post-harvest rot on citrus. Significant viability loss against these fungi was observed with concentrations as low as 1 µM with the compounds citral, carvacrol, and linalool (natural compounds that are safe and readily available from commercial sources). Significant bacterial (e.g., Leuconostoc mesenteroides, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli) viability loss was obtained at concentrations as low as 0.4 µM of these compounds. Data from these in vitro tests will determine which compounds will be used in future testing in (1) the small scale corn silo model; (2) prevention of Leuconostoc mesenteroides in raw sugar cane juice; and (3) safe, food grade edible films, respectively. In FY 2012, research using a small model of stored corn showed that volatilized compound trans-2-hexenal (produced by soybeans when infected with A. flavus) intermittently pumped into non-sterile, wet, whole corn inoculated with A. flavus prevented the growth of this fungus and subsequent aflatoxin production. Results obtained from the study of the antifungal properties of blue light (470 nm). Experiments showed that blue light inhibits the growth of fungi naturally present on whole corn as well as A. flavus inoculated onto the corn kernels.

4. Accomplishments
1. Detection of unique volatiles produced by toxigenic Aspergillus (A.) flavus could be used to protect stored corn from fungal growth. Aspergillus flavus, commonly found on corn, produce aflatoxins (the most potent natural liver cancer-causing compounds known). When conditions are conducive, this fungus readily grows on stored corn and renders it unsafe. ARS scientists in the Food and Feed Safety Research Unit, Southern Regional Research Center, New Orleans, LA, in FY 2012 completed a multi-year effort to identify unique volatiles produced by toxigenic A. flavus isolates on both sterile and non-sterile cracked corn. The first study identified the volatiles produced under optimal conditions when this fungus is grown alone on sterile corn. Later research determined the volatile profile of the A. flavus isolates when grown on non-sterile corn containing naturally-occurring bacteria and fungi. Unique volatile compounds consisted of alcohols, aldehydes, alkanes, terpenes, and a few other chemical families. The number and identity of the specific volatiles were dependent on the toxigenic isolate tested. Our stakeholder, Sensor Development Corporation, used this data to modify a sensor under development and undergoing Beta testing (testing outside lab in real conditions). In late 2012 or early 2013, work will begin on the identification of volatiles produced by Fusarium verticillioides, another toxin-producing fungus commonly found on corn. Detection of these volatiles could be used by companies to remove the contaminated corn batch before it contaminates wholesome corn, thus increasing the supply of wholesome corn for national and international use.

2. Safe, effective, and inexpensive plant volatiles prevent microbial, especially toxigenic fungal, growth and toxin production in a stored corn model. Improperly stored corn can become wet resulting in the growth of toxin-producing fungi (e.g., Aspergillus (A.) flavus, Fusarium verticillioides) that render the corn unsafe for consumption. Trans-2-hexenal (T2H) is a natural antifungal soybean volatile whose production is induced during infection by A. flavus. In FY 2011, ARS scientists in the Food and Feed Safety Research Unit, Southern Regional Research Center, New Orleans, LA, found that T2H in the liquid and volatile forms was highly effective against A. flavus both in vitro and when applied to wet, sterile corn in small models of stored corn. During FY 2012, the in vivo study was expanded where the volatile T2H was intermittently pumped (30 min per two hour or 12 hour cycle over 1-7 seven days) in a large jar containing wet, non-sterile corn. Results showed that this volatile as compared to the control samples, no fungal growth occurred 24 hours after incubation began. The volatile T2H did not remain in the corn after it was removed from the container, indicating that residual T2H would not be a problem if used as a fumigant. This research could aid grain (especially corn) storage companies to protect stored grains from the growth of naturally-occurring fungi and contamination with mycotoxins.

3. In vitro research on the antifungal and antibacterial properties of safe, commercially available plant compounds to determine which are suitable candidates to protect post-harvest food and feed. It is important to prevent the growth of Aspergillus (A.) flavus and Fusarium (F.) verticillioides which are naturally present on corn and produce dangerous toxins that render the corn unsafe for use as food or feed. In vitro testing continued to discover the most active, safe commercially inexpensive plant compounds with activity against these fungi. Examples include citral, found in lemon oil and used in perfumes and flavoring, which kills nearly 100% of A. flavus and F. verticillioides at 0.75 and 2 µM, respectively. Other natural compounds, linalool and carvacrol, that are known antimicrobial plant compounds were not as effective against these fungi. Investigations began on the elimination of Leuconostoc mesenteroides from raw sugarcane juice, and studies continued to find compounds that could be used either as a volatile or in food grade films to protect citrus from post-harvest rot caused by the fungi Penicillium (P.) digitatum and P. italicum. These compounds were also tested against bacteria such as Escherichia coli. ARS scientists in the Food and Feed Safety Research Unit, Southern Regional Research Center, New Orleans, LA, found that citral (5 µM) within 24 hr killed approximately 100% L. mesenteroides inoculated into raw, sterile sugarcane juice. Carvacrol, another natural antimicrobial compound, significantly decreased Leuconostoc (L.) mesenteroides viability, but at a higher concentration than that seen with citral. It is expected that successful research will provide the sugar industry with a safe, natural compound at low concentrations that effectively prevents L. mesenteroides growth and subsequent dextran formation and sucrose loss.

4. Visible blue light (470 nm) is antimicrobial with potential use in food protection. Ultraviolet light (wavelength 10-400 nm) has antimicrobial properties but is a Group 1 carcinogen which causes cancer in humans. However, visible blue light (450-475 nm) is not carcinogenic and has antibacterial properties. ARS scientists in the Food and Feed Safety Research Unit, Southern Regional Research Center, New Orleans, LA, conducted experiments over five days showing blue light light-emitting diodes (LEDs) (470 nm), in contrast to corn kernels not exposed to the LEDs, inhibit the growth of naturally occurring fungi as well as the growth of Aspergillus (A.) flavus inoculated onto the kernels. These results indicate that light of this wavelength could be used to prevent the growth of such fungi on corn while the grain is being moved on conveyor belts. Blue light could also be used to prevent fungal growth on finished baked goods or other products where fungal growth can render the foods inedible. ARS scientists in the Food and Feed Safety Research Unit, Southern Regional Research Center, New Orleans, LA, completed a collaborative study with the University of New Orleans on the antifungal properties of FD&C # 3 red food color (a photosensitive compound) analogs in the presence of blue light. Data showed that the over 25 new analogs produced by the university were not as effective as photosensitizers as the parent compound in the presence of blue light. Results therefore show that the currently used food color, but not the novel analogs, could be used in the presence of blue light (peak: 470 nm) to prevent spoilage fungi from growing on baked goods.